EP3143146B1 - Adenoassociated virus vectors for the treatment of lysosomal storage disorders - Google Patents

Adenoassociated virus vectors for the treatment of lysosomal storage disorders Download PDF

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EP3143146B1
EP3143146B1 EP15735634.6A EP15735634A EP3143146B1 EP 3143146 B1 EP3143146 B1 EP 3143146B1 EP 15735634 A EP15735634 A EP 15735634A EP 3143146 B1 EP3143146 B1 EP 3143146B1
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cag
seq
acetylglucosaminidase
alpha
aav9
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EP3143146A1 (en
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M Fàtima BOSCH TUBERT
M Virginia HAURIGOT MENDOÇA
Albert RIBERA SANCHEZ
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Universitat Autonoma de Barcelona UAB
Esteve Pharmaceuticals SA
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Esteve Pharmaceuticals SA
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Definitions

  • the present invention relates to vectors useful for the expression of proteins of interest and their utilization in gene therapy.
  • the present invention also relates to vectors and nucleic acid sequences helpful for the treatment of mucopolysaccharidoses (MPS), and in particular, for the treatment of mucopolysaccharidoses type III-B or Sanfilippo B syndrome.
  • MPS mucopolysaccharidoses
  • the lysosome is an organelle found in the cytoplasm of animal cells that contains more than 50 hydrolases that break down biomolecules during the recycling of worn-out cellular components or after the engulfment of viruses and bacteria.
  • This organelle contains several types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All enzymes are acid hydrolases.
  • Lysosomal storage diseases are caused by genetic defects that affect one or more lysosomal enzymes. These genetic diseases result generally from a deficiency in a particular enzyme activity present in the lysosome. To a lesser extent, these diseases may be due to deficiencies in proteins involved in lysosomal biogenesis.
  • LSDs are individually rare, although as a group these disorders are relatively common in the general population.
  • the combined prevalence of LSDs is approximately 1 per 5,000 live births. See Meikle P, et al., JAMA 1999;281:249-254 .
  • some groups within the general population are particularly afflicted by a high occurrence of LSDs.
  • the prevalence of Gaucher and Tay-Sachs diseases in descendants from Jewish Central and Eastern European (Ashkenazi) individuals is 1 per 600 and 1 per 3,900 births, respectively.
  • Type III mucopolysaccharidoses known collectively as Sanfilippo syndrome, are LSDs caused by deficiency in one of the enzymes involved in the stepwise degradation of the glycosaminoglycan (GAG) heparan sulfate (HS), leading to its pathological accumulation. MPSIII is classified into four subtypes depending on the enzyme deficiency. Loss of N-acetylglucosaminidase, alpha enzymatic activity causes subtype IIIB. The resulting disease is characterized clinically as a childhood-onset progressive neuropathy of the CNS. The clinical course can generally be divided into three phases.
  • Substrate deprivation therapy aims at reducing the rate of GAG synthesis, so that, if any residual enzymatic activity remains, excessive accumulation of GAGs is prevented or at least the rate of accumulation is slowed down.
  • Genistein a soybean isoflavone, has been suggested to act as an inhibitor of HS production by decreasing the kinase activity of the Epidermal Growth Factor receptor (EGFR). See Jakobkiewicz-Banecka et al., J Biomed Sci. 2009;16:26 , Piotrowska et al., Eur J Hum Genet. 2006;14(7):846-52 .
  • Rhodamine B Another molecule, rhodamine B, has also proven to be effective in decreasing GAG accumulation in preclinical studies, with an efficacy similar to that observed with genistein. See Hendriksz et al., "Guidelines for the investigation and Management of Mucopolysaccharidosis type III", 2012, available at www.mpssociety.co.uk . Rhodamine B is thought to suppress the synthesis of GAG chains by inhibiting the formation of sugar precursors and/or the activity of glycosyl transferases. See Roberts et al., Mol Genet Metab. 2007;92(1-2):115-21 .
  • M6P mannose-6-phosphate
  • ERT enzyme replacement therapy
  • Hematopoietic stem cell transplantation using bone marrow-derived stem cells (Bone marrow transplantation, BMT) has proven efficient in the treatment of both somatic and neurological pathology in patients with other MPSs See Peters et al., Blood 1996;87(11):4894-902 , Peters and Steward, Bone Marrow Transplant 2003;31(4):229-39 and Yamada et al., Bone Marrow Transplant 1998;21(6):629-34 .
  • Donor-derived myeloid cells are able to cross the BBB, enter the brain parenchyma, and differentiate into microglial cells that secrete the missing lysosomal enzyme, which is then taken up by surrounding cells leading to correction of GAG accumulation in the brain.
  • BBB Backbone BB
  • Donor-derived myeloid cells are able to cross the BBB, enter the brain parenchyma, and differentiate into microglial cells that secrete the missing lysosomal enzyme, which is then taken up by surrounding cells leading to correction of GAG accumulation in the brain.
  • HSCT using umbilical cord blood-derived stem cells improved the cognitive outcome in MPSIIIB mice but required repeated cell administration. See Willing et al., Cell Transplantation 2013 ;epub ahead of print. This approach has recently been used to transplant several children with MPSIIIA and MPSIIIB; it is yet unclear whether it results in protection of the CNS from degeneration. See de Ruijter et al., Curr Pharm Biotechnol. 2011;12(6):923-30 .
  • Lentiviral vectors coding for the human N-acetylglucosaminidase, alpha gene have been administered intravenously to young MPSIIIB mice, resulting in low levels of transgene expression in liver, spleen, lung and heart, which reduced but did not normalize GAG accumulation in these tissues. See Di Natale et al., Biochem J. 2005;388(2):639-46 .
  • the therapeutic potential of lentiviral vectors has also been tested by direct delivery of vectors to the brain parenchyma via intracranial administration. See Di Domenico et al., Am J Med Genet A. 2009;149A(6):1209-18 .
  • MPSIIIB mice administered at a single brain site showed increased N-acetylglucosaminidase, alpha activity up to 6 months after treatment, which only partially corrected lysosomal storage lesions.
  • Adenoassociated virus (AAV) vector-mediated gene transfer in particular, is rapidly emerging as the approach of choice for many in vivo gene therapy applications, due to the high transduction efficiency and the lack of pathogenicity of these vectors.
  • AAV vectors can transduce post-mitotic cells and several preclinical and clinical studies have demonstrated the potential of AAV vector-mediated gene transfer to efficiently drive sustained expression of therapeutic transgenes for a variety of diseases. See Bainbridge et al., N Engl J Med. 2008;358(21):2231-9 , Hauswirth et al., Hum Gene Ther. 2008;19(10):979-90 , Maguire et al., N Engl J Med.
  • CSF cerebrospinal fluid
  • a single administration of AAV2 vectors to the cerebrospinal fluid (CSF) by intracisternal injection led to restoration of N-acetylglucosaminidase, alpha activity and reduction of GAGs in the MPSIIIB mouse brain, although no detectable N-acetylglucosaminidase, alpha activity was observed in somatic tissues.
  • AAV5 vectors have, however, a low distribution within the brain parenchyma, and the approach requires multiple injections.
  • AAV9 vectors of serotype 9 have been reported for their use in the treatment of MPSIIIA, which comprise a CAG promoter and codon optimized nucleotide sequences for human sulfamidase expression ( WO2011154520 ), to date, only one study that uses AAV9 vectors for the treatment of MPSIIIB has been reported.
  • the approach takes advantage of the ability of AAV9 vectors to transduce the CNS when systemically administered. See Foust et al., Nat Biotechnol. 2009;27(1):59-65 and Duque, et al., Mol Ther. 2009;17(7):1187-1196 .
  • the present invention provides new recombinant vectors for the treatment of diseases, in particular for the treatment of mucopolysaccharidoses type III (MPSIII), especially MPSIIIB.
  • MPSIII mucopolysaccharidoses type III
  • the invention in a first aspect, relates to a recombinant adenoassociated virus vector serotype 9 (AAV9 vector) containing a CAG promoter SEQ ID NO: 4 linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha (Naglu) SEQ ID NO: 1, wherein the nucleotide sequence coding for Naglu SEQ ID NO: 1 is sequence SEQ ID NO: 3, sequence SEQ ID NO: 19 or sequence SEQ ID NO: 22.
  • the AAV vectors according to the invention are of serotype 9 (AAV9). These vectors proved to be very efficient to fully revert the pathological GAGs storage in all regions of the brain and somatic tissues.
  • the AAV9 vectors of the present invention further contain a promoter linked to the coding nucleotide sequence in order to control the expression of Naglu.
  • the suitable promoter is the CAG promoter, SEQ ID NO: 4.
  • Another aspect of the invention relates to plasmids containing a CAG promoter linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha (Naglu), SEQ ID NO: 1, wherein the CAG promoter and the nucleotide sequence are flanked by AAV2 ITRs, and wherein said plasmids are pAAV-CAG-cohNaglu with accession number DSM 26626, containing the nucleotide sequence SEQ ID NO: 3 coding for N-acetylglucosaminidase, alpha, SEQ ID NO: 1, pAAV-CAG-cohNaglu-version2 with accession number DSM 32042, containing the nucleotide sequence SEQ ID NO: 19 coding for N-acetylglucosaminidase, alpha, SEQ ID NO: 1 or pAAV-CAG-cohNaglu-version3 with accession number DSM 320
  • a further aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the AAV9 vector of the invention or a plasmid described therein.
  • the present description further provides methods useful for delivering polynucleotides, especially Naglu polynucleotides.
  • Still a further aspect of the invention relates to the AAV9 vector of the invention or a plasmid described therein for use as a medicament, in particular for the treatment of mucopolysaccharidoses type III (MPSIII), especially MPSIIIB.
  • MPSIII mucopolysaccharidoses type III
  • the present invention also provides method for the production of the AAV 9 vector according to the invention.
  • the invention relates to isolated cells comprising the AAV9 vector of the invention.
  • the plasmid pAAV-CAG-cohNaglu (SEQ ID NO: 6) was deposited on November 13 th , 2012, under access number DSM 26626 at the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffen No 7 B, D-38124 Braunschweig, Federal Republic of Germany.
  • the plasmid pAAV-CAG-hNaglu (SEQ ID NO: 5) was deposited on March 13 th , 2014, under access number DSM 28568 at the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffen No 7 B, D-38124 Braunschweig, Federal Republic of Germany.
  • the plasmid pAAV-CAG-cohNaglu-version2 (SEQ ID NO: 20) was deposited on April 29 th , 2015, under access number DSM 32042 at the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffen No 7 B, D-38124 Braunschweig, Federal Republic of Germany.
  • the plasmid pAAV-CAG-cohNaglu-version3 (SEQ ID NO: 23) was deposited on April 29 th , 2015, under access number DSM 32043 at the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Inhoffen No 7 B, D-38124 Braunschweig, Federal Republic of Germany.
  • nucleotide sequence refers to a nucleic acid molecule, either DNA or RNA, containing deoxyribonucleotides or ribonucleotides respectively.
  • the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence.
  • % sequence identity refers to the percentage of nucleotides of a candidate sequence that are identical to the nucleotides in the sequence of reference, after aligning the sequences to achieve the maximum % sequence identity.
  • the % sequence identity can be determined by any methods or algorithms established in the art, such as the ALIGN, BLAST and BLAST 2.0 algorithms. See Altschul S, et al., Nuc Acids Res. 1977;25:3389-3402 and Altschul S, et al., J Mol Biol. 1990;215:403-410 .
  • the % sequence identity is calculated dividing the number of nucleotides that are identical after aligning the sequence of reference and the candidate sequence, by the total number of nucleotides in the sequence of reference and multiplying the result by 100.
  • codify or "coding” refer to the genetic code that determines how a nucleotide sequence is translated into a polypeptide or a protein.
  • the order of the nucleotides in a sequence determines the order of amino acids along a polypeptide or a protein.
  • protein refers to a macromolecule composed of one or more linear chains of amino acids or polypeptides. Proteins can suffer post-translational modifications, like the conversion of a cysteine residue to 3-oxoalanine, glycosylation or metal binding. Glycosilation of a protein is the addition of different carbohydrates that are linked covalently to the amino acid chain.
  • an effective amount refers to an amount of a substance sufficient to achieve the intended purpose.
  • an effective amount of an AAV9 vector to increase N-acetylglucosaminidase, alpha (Naglu) activity is an amount sufficient to reduce glycosaminoglycan accumulation.
  • a "therapeutically effective amount” of an expression vector to treat a disease or disorder is an amount of the expression vector sufficient to reduce or eradicate the signs and symptoms of the disease or disorder.
  • the effective amount of a given substance will vary with factors such as the nature of the substance, the route of administration, the size and species of the animal to receive the substance and the purpose of giving the substance. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.
  • the term "individual” refers to a mammal, preferably human or non-human mammal, more preferably mouse, rat, other rodents, rabbit, dog, cat, pig, cow, horse or primate, further more preferably human.
  • operably linked refers to the functional relation and the location of the promoter sequence with respect to the gene of interest (e.g. a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence).
  • a promoter operably linked is contiguous to the sequence of interest.
  • an enhancer does not have to be contiguous to the sequence of interest to control its expression.
  • tropism refers to the way in which different viruses have evolved to preferentially target specific host species, or specific cell types within those species.
  • gene therapy refers to the transfer of genetic material (e.g. DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition.
  • the genetic material of interest encodes a product (e.g. a protein polypeptide, peptide or functional RNA) whose production in vivo is desired.
  • the genetic material of interest can encode an enzyme, hormone, receptor, or polypeptide of therapeutic value.
  • recombinant viral vector refers to an agent obtained from a naturally-occurring virus through genetic engineering techniques capable of transferring genetic material (e.g. DNA or RNA) of interest to a cell, which results in production of the product encoded by that said genetic material (e.g. a protein polypeptide, peptide or functional RNA) in the target cell.
  • genetic material e.g. DNA or RNA
  • a “recombinant vector” or a “vector” is to be understood as being a capsid protein as well as the genetic material contained within used to transfer said genetic material into a cell.
  • a “recombinant vector” or to a “vector” through a nucleotide sequence it means that it refers to a “recombinant vector” or a “vector” whose genome is as set forth in the corresponding sequence listing (SEQ ID).
  • recombinant plasmid refers to a small, circular, double-stranded, self-replicating DNA molecule obtained through genetic engineering techniques capable of transferring genetic material of interest to a cell, which results in production of the product encoded by that said genetic material (e.g. a protein polypeptide, peptide or functional RNA) in the target cell.
  • the term “recombinant plasmid” or “plasmid” also refers to a small, circular, double-stranded, self-replicating DNA molecule obtained through genetic engineering techniques used during the manufacturing of viral vectors as carriers of the recombinant vector genome.
  • the present invention provides new recombinant vectors for the treatment of diseases, in particular for the treatment of mucopolysaccharidoses type III (MPSIII), especially MPSIIIB.
  • MPSIII mucopolysaccharidoses type III
  • the recombinant vector may also contain different functional elements that include control elements for transcription like promoters or operators, transcription factors binding regions or enhancers and control elements for the initiation or termination of translation.
  • the vectors according to the invention are adenoassociated vectors (AAV) that are used to transfer the gene of interest. They have proved to have a high efficiency in transducing post-mitotic cells in wide range of tissue.
  • the vectors are used to deliver a codon optimized human N-acetylglucosaminidase, alpha (cohNaglu) polynucleotide (SEQ ID NO: 3, SEQ ID NO: 19 or SEQ ID NO: 22).
  • An adenoassociated vector is a vector derived from the genome of an adenoassociated virus of the family of parvoviridae. The adenoassociated virus genome is built of single-stranded deoxyribonucleic acid (ssDNA).
  • the serotype is the classification of the viruses groups, depending on their capsid antigens.
  • the serotype of adenoassciated virus determined by its capsid protein, defines the virus tropism and allows its entry into a specific cell type.
  • the serotype 9 of the adenoassociated virus vectors shows the best ability to deliver the genetic material to the brain as well as to peripheral organs upon a single administration.
  • the inventors have surprisingly found that the association, in the same entity, of the AAV9 capsid with a nucleotide sequence coding for the N-acetylglucosaminidase, alpha, together with a specific CAG promoter allows a long-lasting expression of the missing enzyme in all areas of the brain.
  • the lysosomal accumulation of glycosaminoglycan (GAG) is corrected, preventing by that way the neurological alterations characteristic of the MSPIII diseases, and in particular of the MPSIIIB. This effect has been obtained even in the olfactory bulb, which is distant form the point of administration of the vectors.
  • AAV9 vectors according to the invention delivered into the cerebrospinal fluid were able to reach the systemic circulation to transduce the liver.
  • the present invention relates to recombinant AAV9 vectors containing a CAG promoter (SEQ ID NO: 4) linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1, wherein the nucleotide sequence coding for N-acetylglucosaminidase, alpha, SEQ ID NO: 1, is sequence SEQ ID NO: 3, sequence SEQ ID NO: 19 or sequence SEQ ID NO: 22.
  • nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 2.
  • the AAV9 vectors according to the present invention contain a promoter that control the translation and transcription of the gene of interest.
  • a promoter is a nucleotide sequence operably linked to said gene of interest.
  • the promoter used in the present invention is the CAG promoter which refers to the combination comprising the cytomegalovirus early enhancer element and the chicken ⁇ -actin promoter. It further includes a portion of ⁇ -globin intron that confers stability to the mRNA derived from the gene of interest, See Alexopoulou A, et al., BMC Cell Biology 2008; 9(2): 1-11 .
  • the CAG promoter included in the AAV9 vectors of the present invention has a sequence SEQ ID NO: 4. In particular this CAG promoter proved to be more efficient than the CMV promoter usually used in the art.
  • the recombinant AAV9 vector is chosen from AAV9-CAG-hNaglu (SEQ ID NO: 9), AAV9-CAG-cohNaglu (SEQ ID NO: 10), AAV9-CAG-cohNaglu-version2 (SEQ ID NO: 21) and AAV9-CAG-cohNaglu-version3 (SEQ ID NO: 24).
  • the recombinant AAV9 vector is chosen from AAV9-CAG-hNaglu (SEQ ID NO: 9) or AAV9-CAG-cohNaglu (SEQ ID NO: 10).
  • the recombinant AAV9 vectors of the present invention are composed of the viral capsid of the serotype 9 of human adenoassociated virus and a modified genome containing the Inverted Terminal Repeats (ITRs) of human adenoassociated virus serotype 2, the CAG promoter, the Coding Sequence (CDS) of the human alpha N-acetylglucosamidinase (Naglu) gene and the polyA from the rabbit beta-globin gene.
  • ITRs Inverted Terminal Repeats
  • CDS Coding Sequence
  • the present invention also relates to plasmids that contain a CAG promoter linked to a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1, wherein the CAG promoter and the nucleotide sequence are flanked by AAV2 ITRs.
  • the plasmids according to the present invention contain a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that is SEQ ID NO: 3, SEQ ID NO: 19 or SEQ ID NO: 22.
  • plasmids are useful to produce the recombinant AAV9 vectors of the present invention by transfection of HEK293 cells using methods known in the state of the art.
  • nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 2.
  • nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 3.
  • nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 19.
  • nucleotide sequence contained in the plasmids of the invention and coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 is SEQ ID NO: 22.
  • the plasmids of the description are chosen from pAAV-CAG-hNaglu (SEQ ID NO: 5), pAAV-CAG-cohNaglu (SEQ ID NO: 6), pAAV-CAG-cohNaglu-version2 (SEQ ID NO: 20) and pAAV-CAG-cohNaglu-version3 (SEQ ID NO: 23), and especially chosen from pAAV-CAG-hNaglu (SEQ ID NO: 5) and pAAV-CAG-cohNaglu (SEQ ID NO: 6), and preferably the plasmid is pAAV-CAG-cohNaglu (SEQ ID NO: 6).
  • the plasmids of the invention are chosen from pAAV-CAG-cohNaglu (SEQ ID NO: 6) with accession number DSM 26626, containing the nucleotide sequence SEQ ID NO: 3 coding for N-acetylglucosaminidase, alpha, SEQ ID NO: 1, pAAV-CAG-cohNaglu-version2 (SEQ ID NO: 20) with accession number DSM 32042, containing the nucleotide sequence SEQ ID NO: 19 coding for N-acetylglucosaminidase, alpha, SEQ ID NO: 1 or pAAV-CAG-cohNaglu-version3 (SEQ ID NO: 23) with accession number DSM 32043, containing the nucleotide sequence SEQ ID NO: 22 coding for N-acetylglucosaminidase, alpha, SEQ ID NO: 1, and preferably the plasmid is pAAV-CAG-coh
  • the present invention further provides a method for the production of the adenoassociated viral recombinant vectors AAV9 according to the invention.
  • the process comprises the steps of:
  • the AAV first and second terminal repeats of the first vector are ITRs from the AAV serotype 2.
  • the AAV rep genes of the second vector are from the AAV serotype 2.
  • the competent cells are HEK293 cells.
  • the description also provides a method for the preparation of the plasmids according to the invention, comprising the step of:
  • compositions comprising a therapeutically effective amount of the AAV9 vectors of the invention, or a therapeutically effective amount of the plasmids of the invention.
  • compositions of the invention comprise the recombinant AAV9 vectors in a pharmaceutically acceptable carrier.
  • the composition may also comprise at least one auxiliary substance.
  • the auxiliary substances can be selected among carriers, excipients, solvents, diluents, or adjuvants.
  • Acceptable carriers, diluent or adjuvants are non-toxic and are preferably inert at the dosage and concentrations employed and include buffers such as phosphate, citrate or other organic acids; antioxidants; low molecular weight polypeptides, proteins such as serum albumin, gelatin or immunoglobulins; hydriophilic polymers; aminoacids; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents; sugar alcohols such as mannitol or sorbitol, salt forming couterions such as sodium; and/or non-ionic surfactants such as polyethylene-polyoxypropylene block copolymer (Pluronic F68®) polyethylene glycol (PEG).
  • buffers such as phosphate, citrate or other organic acids
  • antioxidants low molecular weight polypeptides, proteins such as serum albumin, gelatin or immunoglobulins
  • hydriophilic polymers aminoacids
  • the pharmaceutical compositions according to the invention are suitable for parenteral administration.
  • parenteral administration are intravenous, subcutaneous, intracisternal and intramuscular injections.
  • the pharmaceutical composition according to the invention is suitable for intravenous or intracisternal administration.
  • Compositions suitable for such parenteral administration include sterile aqueous solutions or dispersions, sterile powders for extemporaneous preparation of sterile solutions or dispersions.
  • the pharmaceutical compositions according to the invention are preserved from contaminating action of bacteria and fungi.
  • the daily dosage for humans and animals may vary depending on factors that have their basis in the respective species or other factors, such as age, sex, weight or degree of illness and so forth.
  • Another aspect of the present invention relates to the therapeutical use of the AAV9 vectors described hereinbefore, or the plasmids described hereinbefore.
  • the recombinant AAV9 vectors according to the invention achieve an expression of the missing Naglu enzyme, thus correcting the lysosomal accumulation of GAGs. This allows correcting all clinical signs of the mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB.
  • the present invention also concerns the recombinant AAV9 vectors or the plasmids of the invention for use as a medicament.
  • the invention relates to the recombinant AAV9 vectors or the plasmids of the invention for increasing the alpha N-glucosaminidase activity in the body for use in the treatment of mucopolysaccharidoses.
  • the present invention relates to the recombinant AAV9 vectors or the plasmids of the invention for use in the treatment of mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB.
  • MPSIII mucopolysaccharidoses type III
  • the present description relates to the use of the recombinant AAV9 vectors described hereinbefore, or the plasmids described hereinbefore for the manufacture of a medicament useful for the treatment of mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB.
  • MPSIII mucopolysaccharidoses type III
  • Another embodiment of the present description is directed to the method of treatment of mucopolysaccharidoses type III (MPSIII) and especially MPSIIIB, comprising the step of administering at least a recombinant AAV9 vector described hereinbefore, or at least a plasmid described hereinbefore to a subject in need thereof.
  • MPSIII mucopolysaccharidoses type III
  • the present description further provides an isolated cell comprising the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
  • the cell according to the description comprises a nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 that has at least 80 % sequence identity to SEQ ID NO: 2.
  • the nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has at least 84 % sequence identity to SEQ ID NO: 2.
  • nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has a 84 %, 87 %, 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 2.
  • nucleotide sequence coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1 has at least 85 % sequence identity to SEQ ID NO: 3, and preferably 85 %, 87 %, 90 %, 95 %, 98 %, or 99 % sequence identity to SEQ ID NO: 3.
  • the cells of the description comprise the nucleotide sequence SEQ ID NO: 2 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
  • the cells of the description comprise the nucleotide sequence SEQ ID NO: 3 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
  • the cells of the description comprise the nucleotide sequence SEQ ID NO: 19 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
  • the cells of the description comprise the nucleotide sequence SEQ ID NO: 22 coding for N-acetylglucosaminidase, alpha SEQ ID NO: 1.
  • Another aspect of the invention refers to an isolated cell comprising any of the recombinant AAV9 vectors of the invention.
  • the AAV vectors described herein were obtained by triple transfection.
  • the materials required for making the vectors were: HEK293 cells (expressing E1 genes), helper plasmid providing adenovirus function, plasmid providing AAV rep genes from serotype 2 and cap genes from the serotype 9 (AAV9) and, finally, the backbone plasmid with AAV2 ITRs and the construct of interest.
  • alpha-expressing AAV vectors To generate N-acetylglucosaminidase, alpha-expressing AAV vectors, the non-optimized or optimized CDS of human, murine or canine N-acetylglucosaminidase, alpha were cloned into an AAV backbone plasmid under the control of the ubiquitous hybrid CAG promoter.
  • Vectors were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita T, et al., Gene Ther. 1998;5:938-945 and Wright J, et al., Mol. Ther. 2005;12:171-178 .
  • Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, NY, US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by the viral ITRs of serotype 2 AAV (described above); 2) a plasmid carrying the AAV rep2 and the cap9 genes; and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using an optimized protocol as previously described. See Ayuso E, et al., Gene Ther. 2010;17:503-510 . Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • the vectors of the present invention were constructed according to molecular biology techniques well known in the art.
  • a congenic mutant C57B1/6J N-acetylglucosaminidase, alpha-deficient mouse (MPSIIIB) model was purchased from The Jackson Laboratory (Bar Harbor, ME, USA. Stock 003827). See Li et al., Proc Natl Acad Sci. 1999; 96(25):14505-10 . Affected MPSIIIB and healthy control mice were inbred from heterozygous founders. Genotype was determined on genomic DNA from tail-clipped samples with a PCR analysis that amplifies a sequence encompassing the targeted mutation.
  • the sequences of the respective sense and antisense primers were: Forward Primer: 5'-GTC GTC TCC TGG TTC TGG AC-3' (SEQ ID NO: 13), Reverse Primer: 5'-ACC ACT TCA TTC TGG CCA AT-3' (SEQ ID NO: 14), Reverse Primer Mutation: 5'- CGC TTT CTG GGC TCA GAG-3' (SEQ ID NO: 15). Mice were fed ad libitum with a standard diet (Harlan, Tekland)) and maintained under a light-dark cycle of 12 h (lights on at 9:00 A.M.).
  • mice For intracisternal delivery of AAV9-CAG-comNaglu vectors to mice, a total dose of 3x10 10 vg were injected to the cisterna magna of 2-month-old MPSIIIB animals. A similar cohort of animals was injected with 3.9x10 10 vg control non-coding (AAV9-null) vector. At 5 months of age, i.e. 3 months post vector administration, mice were anesthetized and then transcardially perfused with 10 ml of PBS to completely clear blood from tissues.
  • the entire brain and multiple somatic tissues were collected and either frozen in liquid nitrogen and stored at -80°C or immersed in formalin for subsequent histological analyses.
  • mice were sacrificed and organs were harvested as described in the previous paragraph.
  • AAV9-CAG-cocNaglu vectors For intracisternal delivery of AAV9-CAG-cocNaglu vectors to dogs, a total dose of 6.5x10 12 vg was administered to healthy adult Beagle dogs via cisterna magna injection. Two of the animals received an intravenous injection of 1x10 11 vg/kg of AAV9-null vectors 6 weeks prior to administration of Naglu vectors to pre-immunize them against AAV9. First weekly, and then monthly, CSF and serum samples were collected and stored at -80°C.
  • Tissues ( ⁇ 100 mg) were digested overnight (ON) at 56°C in 400 ⁇ l of Proteinase K solution (0.2 mg/ml).
  • Total DNA was isolated from supernatants by extraction using standard techniques. DNA was resuspended in distilled water and quantified using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). Vector genome copy number in 20 ng of total DNA was determined by quantitative real time PCR with primers and probe specifics for the murine N-acetylgluosaminidase, alpha transgene that do not amplify the endogenous genomic locus.
  • glycosaminoglycan For glycosaminoglycan (GAG) quantification, tissue samples were weighted and then digested with proteinase K and extracts were clarified by centrifugation and filtration. GAG levels were determined in tissue extracts with the Blyscan sulfated glycosaminoglycan kit (Biocolor, Carrickfergus, County Antrim, GB), using chondroitin 4-sulfate as standard. The levels of GAG were normalized to wet tissue weight.
  • Blyscan sulfated glycosaminoglycan kit Biocolor, Carrickfergus, County Antrim, GB
  • IDUA activity was measured in 15 ⁇ g of protein incubated for 1 h at 37°C with 4-methylumbelliferyl ⁇ -N-iduronide (Glycosynth).
  • IDS activity 15 ⁇ g of protein were first incubated with 4-methylumbelliferyl-a-L-iduronide-2-sulphate (Moscerdam Substrates) for 4 h at 37°C, followed by a second 24 h incubation at 37°C with a pool of lysosomal enzymes from bovine testis (LEBT-M2, Moscerdam Substrates).
  • SGSH activity was measured as previously described. See Haurigot et al., J Clin Invest 2013;123(8):3254-71 .
  • GUSB activity 10 ⁇ g of protein were incubated with 4-methylumbelliferyl- ⁇ -D-glucuronide (Sigma) at 37°C for 1 h.
  • HEXB activity was assayed by incubation of 0.1 ⁇ g of protein with 4-methylumbelliferyl N-acetyl- ⁇ -D-glucoaminide (Sigma) for 1 h at 37°C. After stopping reactions by increasing the pH, released fluorescence was measured with FLx800 fluorimeter (BioTek Instruments). All enzyme activities were normalized against total protein content quantified by Bradford (Bio-Rad).
  • Tissues were fixed for 12-24 h in formalin, embedded in paraffin and sectioned.
  • paraffin sections were subjected to heat-induced epitope retrieval in citrate buffer, pH 6, and then incubated overnight at 4°C with rat anti-LAMP1 antibody (1D4B; Santa Cruz Biotechnology, Santa Cruz, CA, US) diluted at 1:100 and subsequently incubated with biotinylated rabbit anti-rat antibody (Dako, Glostrup, DK) at 1:300.
  • paraffin sections were incubated overnight at 4°C with rabbit anti-LIMP2 antibody (NB400; Novus Biologicals, Littleton, CO, USA) diluted at 1:100 and subsequently incubated with biotinylated goat anti-rabbit antibody (31820; Vector Laboratories, Burlingame, CA, USA) at 1:300.
  • rabbit anti-LIMP2 antibody NB400; Novus Biologicals, Littleton, CO, USA
  • biotinylated goat anti-rabbit antibody 31820; Vector Laboratories, Burlingame, CA, USA
  • GFAP immunostaining in brain samples paraffin sections were incubated overnight at 4°C with rabbit anti-GFAP antibody (Ab6673; Abcam, Cambridge, UK) diluted at 1:1000 and subsequently incubated with biotinylated goat anti-rabbit antibody (31820; Vector Laboratories, Burlingame, CA, USA) at 1:300.
  • LAMP1, LIMP2 and GFAP signals were amplified by incubating sections with ABC-Peroxidase staining kit (Thermo Scientific, Waltham, MA, US) at 1:100 dilution and visualized using 3,3-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, US) as a chromogen. Brightfield images were obtained with an optical microscope (Eclipse 90i; Nikon, Tokyo, JP).
  • Bsi-B4 lectin L5391; Sigma-Aldrich, St. Louis, MO, USA
  • Bsi-B4 signal was visualized using 3,3-diaminobenzidine (Sigma-Aldrich, St. Louis, MO, US) as a chromogen.
  • Brightfield images were obtained with an optical microscope (Eclipse 90i; Nikon, Tokyo, JP).
  • the NIS Elements Advanced Research 2.20 software was used to quantify LIMP2, GFAP, and Bsi-B4 signals in 3-5 images of each brain region (original magnification, ⁇ 20) per animal, using the same signal threshold settings for all animals. Then, the percentage of positive area was calculated, i.e., the area, in pixels, with a positive signal over the total tissue area in the image.
  • mice were sacrificed by an overdose of isofluorane (Isofluo, Labs. Esteve, Barcelona, ES) and perfused via inferior vena cava with 1 ml of 2.5% glutaraldehyde and 2% paraformaldehyde.
  • a small portion (approximately 1mm 3 ) of the lateral lobe of the liver and of the cerebral cortex were sectioned and incubated for 2 hours at 4°C in the same fixative.
  • the specimens were postfixed in 1% osmium tetroxide, stained in aqueous uranyl acetate, and then dehydrated through a graded ethanol series and embedded in epoxy resin. Ultrathin sections (600-800 ⁇ ) from the resin blocks were stained using lead citrate and examined in a transmission electron microscope (H-7000; Hitachi, Tokyo, JP).
  • RNA was isolated with mirVanaTM (Ambion, Life Technolo- gies).
  • cDNA was synthesized and subsequently hybridized in the GeneChip Mouse Gene 2.1 ST 16 array plate (Affymetrix) by Progenika Biopharma (Spain); sample processing, hybridization and scanning were carried out following Affymetrix recommended protocols and equipment.
  • Data normalization was done by RMA (Robust Multiarray averaging) method using Affymetrix® Expression Console TM tool, obtaining log2 transformed normalized values. Data were filtered to focus the analysis on known coding sequences, obtaining an initial list of 26688 altered genes, which were subsequently refiltered to remove genes with variance below the 75th percentile.
  • the human N-acetylglucosaminidase, alpha coding sequence was utilized as starting material (NCBI Reference Sequence: NM_000263) and chemically synthetized for this purpose (GeneArt; Life Technologies).
  • the CDS was received cloned inside the plasmid pMA (AmpR) flanked by MluI and EcoRI restriction sites at 5' and 3' ends, respectively.
  • N-acetylglucosaminidase, alpha CDS was excised by MluI/EcoRI digestion and then cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG (AmpR).
  • the resulting plasmid was named pAAV-CAG-hNaglu (accession number DSM 28568). See SEQ ID NO: 5, and Figure 19 A .
  • the pAAV-CAG plasmid had been previously generated and contained the ITRs from the AAV2 genome, the CAG promoter, and the polyA signal from rabbit ⁇ -globin, as well as a multicloning site for cloning of CDSs of interest.
  • the CAG promoter is a hybrid promoter composed of the CMV early/intermediate enhancer and the chicken ⁇ -actin promoter. This promoter is able to drive a potent expression ubiquitously. See Sawicki J et al., Exper Cell Res. 1998;244:367-369 , Huang J et al., J Gene Med. 2003;5:900-908 , Liu Y et al., Exp Mol Med. 2007; 39(2):170-175 .
  • Vectors AAV9-CAG-hNaglu (SEQ ID NO: 9 and Figure 19 B) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita, 1998, supra and Wright, 2005, supra.
  • Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, NY, US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs (pAAV-CAG-hNaglu); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described. See Ayuso, 2010, supra. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • Expression cassettes including an optimized version of the human N-acetylglucosaminidase, alpha CDS (cohNaglu) were designed and obtained.
  • the sequence optimization (GeneArt®) was performed to maximize the efficiency of N-acetylglucosaminidase, alpha protein production in human beings through elimination of cryptic splice sites and RNA destabilizing sequence elements for increased RNA stability, addition of RNA stabilizing sequence elements, codon optimization and G/C content adaptation, avoidance of stable RNA secondary structures amongst others changes.
  • the optimized CDS was received cloned in the plasmid pMA-RQ (AmpR) flanked by MluI and EcoRI restriction sites at 5' and 3', respectively.
  • the pMA-RQ-cohNaglu plasmid was digested with MluI and EcoRI to excise the optimized N-acetylglucosaminidase, alpha CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-cohNaglu plasmid (accession number DSM 26626). See SEQ ID NO:6 and Figure 20 A .
  • Vectors AAV9-CAG-cohNaglu (SEQ ID NO: 10 and Figure 20 B) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita, 1998, supra and Wright, 2005, supra.
  • Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, NY, US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs (pAAV-CAG-cohNaglu); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described. See Ayuso, 2010, supra. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • Expression cassettes including a second optimized version of the human N-acetylglucosaminidase, alpha CDS (cohNaglu-version2) were designed and obtained.
  • the optimized CDS (DNA 2.0®) was received cloned in the plasmid pJ208 (AmpR) flanked by MluI and EcoRI restriction sites at 5' and 3', respectively.
  • the pJ208-cohNaglu-version2 plasmid was digested with MluI and EcoRI to excise the optimized N-acetylglucosaminidase, alpha-version2 CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-cohNaglu-version2 plasmid (accession number DSM 32042). See SEQ ID NO:20 and Figure 21A .
  • Vectors AAV9-CAG-cohNaglu-version2 (SEQ ID NO: 21 and Figure 21 B) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita, 1998, supra and Wright, 2005, supra.
  • Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, NY, US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs (pAAV-CAG-cohNaglu-version2); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described. See Ayuso, 2010, supra. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • Expression cassettes including a third optimized version of the human N-acetylglucosaminidase, alpha CDS (cohNaglu-version3) were designed and obtained.
  • the optimized CDS (GenScript, Inc) was received cloned in the plasmid pUC57 (AmpR) flanked by MluI and EcoRI restriction sites at 5' and 3', respectively.
  • the pUC57-cohNaglu-version3 plasmid was digested with MluI and EcoRI to excise the optimized N-acetylglucosaminidase, alpha-version3 CDS. Subsequently, this fragment was cloned between the same restriction sites of the pAAV-CAG backbone plasmid to generate the pAAV-CAG-cohNaglu-version3 plasmid (accession number DSM 32043). See SEQ ID NO:23 and Figure 22 A .
  • Vectors AAV9-CAG-cohNaglu-version3 (SEQ ID NO: 24 and Figure 22 B) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita, 1998, supra and Wright, 2005, supra.
  • Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, NY, US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs (pAAV-CAG-cohNaglu-version3); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described. See Ayuso, 2010, supra. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • the CDS for murine N-acetylglucosaminidase, alpha was subjected to sequence optimization (GeneArt; Life Technologies).
  • the optimized CDS was received cloned inside the plasmid pMA-RQ (AmpR) flanked by MluI and EcoRI restriction sites at 5' and 3', respectively.
  • the MluI/EcoRI optimized murine N-acetylglucosaminidase, alpha CDS fragment was excised from the pMA-RQ plasmid and subsequently cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG.
  • the resulting plasmid was named pAAV-CAG-comNaglu. See SEQ ID NO: 7 and Figure 23 A .
  • Vectors AAV9-CAG-comNaglu (SEQ ID NO: 11 and Figure 23 B) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita, 1998, supra and Wright, 2005, supra.
  • Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, NY, US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs (pAAV-CAG-comNaglu); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described. See Ayuso, 2010, supra. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • the CDS for canine N-acetylglucosaminidase, alpha was subjected to sequence optimization (GeneArt; Life Technologies).
  • the optimized CDS was received cloned inside the plasmid pMA-RQ (AmpR) flanked by MluI and EcoRI restriction sites at 5' and 3', respectively.
  • the MluI/EcoRI optimized canine N-acetylglucosaminidase, alpha CDS fragment was excised from the pMA-RQ plasmid and subsequently cloned between the MluI and EcoRI restrictions sites of the AAV backbone plasmid pAAV-CAG.
  • the resulting plasmid was named pAAV-CAG-cocNaglu. See SEQ ID NO: 8 and Figure 24 A .
  • Vectors AAV9-CAG-cocNaglu (SEQ ID NO: 12 and Figure 24 B) were generated by helper virus-free transfection of HEK293 cells using three plasmids with modifications. See Matsushita, 1998, supra and Wright, 2005, supra.
  • Cells were cultured to 70% confluence in roller bottles (RB) (Corning, Corning, NY, US) in DMEM supplemented with 10% FBS and then co-transfected with: 1) a plasmid carrying the expression cassette flanked by AAV2 ITRs (pAAV-CAG-cocNaglu); 2) a plasmid carrying the AAV2 rep and the AAV9 cap genes (pREP2CAP9); and 3) a plasmid carrying the adenovirus helper functions.
  • Vectors were purified by two consecutives cesium chloride gradients using either a standard protocol or an optimized protocol as previously described. See Ayuso, 2010, supra. Vectors were dialyzed against PBS, filtered, titred by qPCR and stored at -80°C until use.
  • a total dose of 50 ⁇ g of the plasmid pAAV9-CAG-hNaglu containing the wild-type human N-acetylglucosaminidase, alpha expressing cassette were administered to 2-month-old MPSIIIB mice via hydrodynamic tail vein (HDTV) injection.
  • This technique targets expression of the delivered plasmid to the liver. See Liu et al., Gene Ther. 1990;6(7): 1258-66 .
  • a total dose of 5x10 11 vector genomes of AAV9-CAG-hNaglu vectors was administered to 2-month-old MPSIIIB mice via tail vein injection.
  • Example 15 Intravenous delivery of AAV9-CAG-cohNaglu
  • a total dose of 5x10 11 vector genomes of AAV9-CAG-cohNaglu vectors was administered to 2-month-old MPSIIIB mice via tail vein injection.
  • N-acetylglucosaminidase Two months after administration, treated animals showed high levels of activity of N-acetylglucosaminidase, alpha in the liver (600 % of healthy levels) and a moderate increase (7% of healthy levels) in the levels of activity in serum.
  • N-acetylglucosaminidase, alpha production completely eliminated or considerably reduced the pathological accumulation of GAGs observed in the somatic tissues of untreated MPSIIIB mice.
  • Figure 4C In addition, treated animals showed significant levels of N-acetylglucosaminidase, alpha activity (18-27% of healthy mice) in all brain regions analysed.
  • Figure 5A This partial restoration of N-acetylglucosaminidase, alpha activity was sufficient to clear GAG storage from all areas of the brain. See Figure 5B .
  • a total dose of 9.3x10 9 vector genomes of AAV9-CAG-hNaglu vector was injected into the cisterna magna of 2-month-old MPSIIIB animals in a total volume of 5 ⁇ l.
  • N-acetylglucosaminidase alpha activity was detected in the liver of treated MPSIIIB mice at levels of 32% of healthy animals. See Figure 7A .
  • This increase in N-acetylglucosaminidase, alpha activity mediated the correction of GAG accumulation in liver, spleen, heart, lung, testis and urinary bladder and also significantly diminished GAG storage in kidney. See Figure 7B .
  • Example 17 Intracisternal delivery of AAV9-CAG-cohNaglu
  • a total dose of 9.3x10 9 vector genomes of AAV9-CAG-cohNaglu vector was injected into the cisterna magna of 2-month-old MPSIIIB animals in a total volume of 5 ⁇ l.
  • Example 18 Hydrodynamic delivery of the plasmid pAAV9-CAG-cohNaglu-version2
  • a total dose of 50 ⁇ g of the plasmid pAAV9-CAG-cohNaglu-version2 carrying an expression cassette containing an optimized version (version2) of human N-acetylglucosaminidase, alpha were administered to 2-month-old MPSIIIB mice via tail hydrodynamic tail vein injection.
  • this technique targets expression of the delivered plasmid to the liver. See Liu et al., supra.
  • N-acetylglucosaminidase alpha activity was increased over pre-treatment levels in the liver and serum of all the animals that received a hydrodynamic injection of the plasmid containing the optimized version2 of N-acetylglucosaminidase, alpha-coding sequence. See Figures 10A and 10B . No activity was detected in MPSIIIB animals injected with saline solution. In treated animals, liver and serum N-acetylglucosaminidase, alpha activity reached levels that were 150% and 1500%, respectively, of the mean value of activity observed in WT animals (set to 100%). See Figures 10A and 10B .
  • Example 19 Hydrodynamic delivery of the plasmid pAAV9-CAG-cohNaglu-version3
  • a total dose of 50 ⁇ g of the plasmid pAAV9-CAG-cohNaglu-version3 containing the a codon optimized version (version3) human N-acetylglucosaminidase, alpha expressing cassette were administered to 2-month-old MPSIIIB mice via tail hydrodynamic tail vein injection.
  • this technique targets expression of the delivered plasmid to the liver. See Liu et al., supra.
  • a total dose of 3x10 10 vector genomes of AAV9-CAG-comNaglu vector was injected into the cisterna magna of 2-month-old MPSIIIB animals in a total volume of 10 ⁇ l.
  • AAV9 vector genomes could be detected in all brain areas analysed, as well as in the spinal cord. In peripheral tissues, vector genomes could be detected at considerable gene copy numbers only in the liver, and at low gene copy numbers in the lymph nodes in which the head drains (mandibular lymph nodes). See Figure 12A-B .
  • IDS Iduronate 2-sulfatase
  • SGSH N-sulphoglucosamine sulphohydrolase
  • GUSB ⁇ -glucuronidase
  • HEXB ⁇ -hexosaminidase
  • CTEN Cell Type Enrichment
  • AAV9 vectors administered to the CSF leak to the periphery and transduce the liver. See Figure 9 and Haurigot et al., supra. Accordingly, an increase in N-acetylglucosaminidase, alpha activity was documented in the liver and serum of MPSIIIB mice treated with AAV9-CAG-comNaglu, reaching levels of approximately 800% of the levels observed in healthy animals. See Figures 16A and 16B . The levels of enzymatic activity in serum correlated well with those of activity in the liver of treated mice, suggesting that the liver was the main source of circulating enzyme.
  • the first step towards the clinical application of a gene therapy approach requires the demonstration of its feasibility in a large animal model.
  • a total dose of 6.5x10 12 vg AAV9-CAG-cocNaglu vectors was administered to the cisterna magna of 4 adult Beagle dogs (Dogs 1-4).
  • Dogs 3 and 4 were immunized by systemic administration of 1x10 11 vg/kg of non-coding AAV9-null vectors 6 weeks before CSF delivery.
  • NAbs anti-AAV9 neutralizing antibodies

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